No abstract

The presence of antibodies to a KSHV antigenic peptide correlates with the presence of Kaposi's sarcoma in a high-risk population and provides further evidence of an etiologic role for KSHV.

Herpesvirus gene expression can be classified into four distinct kinetic stages: latent, immediate early, early, and late. Here we characterize the kinetic class of a group of 16 Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 genes in a cultured primary effusion cell line and examine the expression of a subset of these genes in KS biopsies. Expression of two latent genes, LANA and vFLIP, was constitutive and was not induced by chemicals that induce the lytic cycle in primary effusion lymphoma (PEL) cell lines. An immediate-early gene, Rta (open reading frame 50 [ORF50]), was induced within 4 h of the addition of n-butyrate, and its 3.6-kb mRNA was resistant to inhibition by cycloheximide. Early genes, including K3 and K5 that are homologues of the “immediate-early” gene of bovine herpesvirus 4, K8 that is a positional homologue of Epstein-Barr virus BZLF1, vMIP II, vIL-6, and polyadenylated nuclear (PAN) RNA, appeared 8 to 13 h after chemical induction. A second group of early genes that were slightly delayed in their appearance included viral DHFR, thymidylate synthase, vMIP I, G protein-coupled receptor, K12, vBcl2, and a lytic transcript that overlapped LANA. The transcript of sVCA (ORF65), a late gene whose expression was abolished by Phosphonoacetic acid, an inhibitor of KSHV DNA replication, did not appear until 30 h after induction. Single-cell assays indicated that the induction of lytic cycle transcripts resulted from the recruitment of additional cells into the lytic cycle. In situ hybridization of KS biopsies showed that about 3% of spindle-shaped tumor cells expressed Rta, ORF K8, vIL-6, vMIP I, vBcl-2, PAN RNA, and sVCA. Our study shows that several KSHV-encoded homologues of cellular cytokines, chemokines, and antiapoptotic factors are expressed during the viral lytic cycle in PEL cell lines and in KS biopsies. The lytic cycle of KSHV, probably under the initial control of the KSHV/Rta gene, may directly contribute to tumor pathogenesis.

An Epstein-Barr viral gene (ZEBRA) is identified that, in human lymphoblastoid cells, activates a switch causing the virus to shift from the latent to the replicative phase of its life cycle. We have shown that a 2.7-kilobase-pair rearranged Epstein-Barr virus DNA fragment of this gene (BamHI fragment WZhet) induced transient expression of viral replicative antigens and polypeptides when it was transfected into a somatic cell hybrid, which was derived from the fusion of an epithelial line cell with a Burkitt lymphoma cell. We now show that this rearranged WZhet fragment, when introduced stably into lymphoblastoid cells, will activate expression of the complete viral replicative cycle in 1-10% of the lymphoblastoid cells, leading to production of biologically active virions that can immortalize primary lymphocytes. The transfected plasmid appears to be regulated in a manner analogous to the complete Epstein-Barr virus genome.

The BC-1 cell line, derived from a body cavity-based, B-cell lymphoma, is dually infected with Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In these studies, the relationships between these two gammaherpesviruses and BC-1 cells were characterized and compared. Single-cell cloning experiments suggested that all BC-1 cells contain both genomes. In more than 98% of cells, both viruses were latent. The two viruses could be differentially induced into their lytic cycles by chemicals. EBV was activated into DNA replication and late-gene expression by the phorbol ester tetradecanoyl phorbol acetate (TPA). KSHV was induced into DNA replication and late-gene expression by n-butyrate. Amplification of both EBV and KSHV DNAs was inhibited by phosphonoacetic acid. Induction of the KSHV lytic cycle by n-butyrate was accompanied by the disappearance of host-cell ␤-actin mRNA. Induction of EBV by TPA was not accompanied by such an effect on host-cell gene expression. Induction of the KSHV lytic cycle by n-butyrate was associated with the expression of several novel polypeptides. Recognition of one of these, p40, served as the basis of development of an assay for antibodies to KSHV in the sera of infected patients. BC-1 cells released infectious EBV; however, there was no evidence for the release of encapsidated KSHV genomes by BC-1 cells, even though n-butyratetreated cells contained numerous intranuclear nucleocapsids. The differential inducibility of these two herpesviruses in the same cell line points to the importance of viral factors in the switch from latency to lytic cycle.

Al cells that harbor the Epstein-Barr virus (EBV) genome contain a neoantigen in the nucleus (EBNA). By transfection we located a segment of the genome that encodes or induces an antigen serologically related to EBNA. The responsible genes are found in the 3.4-megadalton BamHI fragment K of EBV DNA, specifically in the left 1.9 megadaltons represented by HindIu fragment I. Mouse LTK-cells were cotransformed with recombinant plasmids containing the herpes simplex virus thymidine kinase gene and either EcoRP fragment B or BamHI fragment K ofEBV DNA. The TKV cells surviving in selective medium were cloned. About 50% of the clones expressed the neoantigen in every nucleus. These mouse cells were used as antigens in immunofluorescence tests. Antibody to the nuclear antigen was found in 30 human sera known to contain antibody to EBNA; it was not detected in 18 sera that did not have antibody to EBNA. Mouse cells expressing EBNA as the result of acquisition of cloned EBV DNA fragments should prove useful in the characterization of the structure of this antigen and as reagents for the diagnosis of EBV infections.Genetic study of the human lymphotropic herpes virus Epstein-Barr virus (EBV) is hindered by the lack of mutants and by the absence ofa fully permissive host cell that allows genetic recombination between viruses. In view of these limitations, a suitable approach for defining the products ofsome EBV genes is gene transfer with defined segments of viral DNA. This approach is feasible because DNA prepared from virions is infectious by either microinjection or transfection (1-3). Human placental fibroblasts exposed to intact virion DNA produce EBV which is able to immortalize lymphocytes (ref. 2; unpublished data). Several different types of tissue culture cells exposed to EBV DNA display various morphologic forms ofantigens in the cytoplasm, nuclear membrane, and nucleus; these antigens are detectable by certain human sera with antibody to EBV (3,4).If a mixture of virion DNA fragments produced by cleavage with one of several restriction endonucleases is introduced, antigens also appear in the fibroblasts. IfEcoRI and Sal I are used, antigens appear in both nucleus and cytoplasm but if BamHI and HindIII, are used, the antigen seems to be limited to the nucleus (4). The need to identify this nuclear antigen and its relationship to "EB nuclear antigen" (EBNA) underlies the present experiments.The finding that a mixture of virion DNA fragments led to nuclear antigen expression suggested the possibility that individual cloned subfragments of viral DNA would also be able to induce the antigen. We found that a single large cloned fragment, EcoRI fragment B of EBV (FF41) DNA, approximately 19 megadaltons, was competent to cause expression ofa nuclear antigen in human fibroblasts (4). We were unable to study this nuclear antigen in human cells by anticomplement immunofluorescence, the usual immunologic assay for EBNA, because of nonspecific binding of complement to the cytoskeleton of the placental fibroblasts. ...

We recently identified, by means of cotransformation of LTK-cells, a region of the Epstein-Barr virus (EBV) genome (the BamHI K fragment) that encodes or induces an EBV nuclear neoantigen (EBNA) serologically related to the EBNA found in lymphoid cells carrying the entire EBV genome. We now find that a second EBV DNA fragment, BamHI M, is also able to give rise to cotransformed LTK-cells with stable expression of a nuclear antigen. The BamHI K and M fragments have no apparent DNA homology. Many human sera that are reactive to EBNA in Raji cells detect both antigens; however, certain anti-EBNA-positive human sera are discordant and react only with the BamHI M or only with the BamHI K nuclear antigen. Every Raji cell appears to express both "M" and "K" antigens; D98 Raji cells, a somatic cell hybrid, express only "K" antigen. The K antigen is found on metaphase chromosomes of LTK cells and Raji cells. The Minduced antigen is not located on chromosomes when the cells are in metaphase but is present as granules within the nucleus.A nuclear neoantigen (EBNA) is found in Epstein-Barr virus (EBV)-associated tumors such as Burkitt lymphoma, polyclonal B-cell lymphoma, and nasopharyngeal cancer (1). EBNA is located on chromosomes in lymphoid cell cultures and in mitotic circulating B lymphocytes in infectious mononucleosis (2). During the process of immortalization of lymphocytes in vitro, EBNA appears within a few hours after infection by the virus (3). Therefore, EBNA is thought to play an important role in the immortalization reaction. The biochemical definition of EBNA is still unclear; a number of different molecular weights and other properties of EBNA have been described (4)(5)(6)(7). By cotransformation of mouse LTK-cells with the herpes simplex thymidine kinase (tk) gene and cloned EBV DNA, we recently mapped one gene that encodes or induces EBNA to the leftmost 2,900 base pairs of the BamHI K fragment of EBV DNA (8). In the course of these studies, we found that LTK-cells also expressed a nuclear antigen transiently after exposure to the BamHI M EBV DNA fragment (about 4.7 kilobase pairs). We have now stably introduced the BamHI M fragment into mouse LTK-cells by cotransformation. The nuclear antigen (M) that such cells express is also an EBNA but is serologically distinct from the BamHI K nuclear antigen. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

The sera of 12 patients with presumed chronic active Epstein-Barr virus (EBV) infection lacked antibody to a component of the Epstein-Barr nuclear antigen (EBNA) complex encoded by the BamHI K fragment of viral DNA. This anomaly, detected in approximately 18% of sera obtained from patients with a diagnosis of "chronic mononucleosis," was more often found in patients with severe disease (approximately 32%) who had objective clinical findings and markedly elevated antibody titers to EBV replicative antigens than in those patients with the "fatigue syndrome" (10%). The lack of antibody to the K nuclear antigen is specific because most of those who did not have antibody to the K antigen made antibody to other latent nuclear (EBNA 2) antigens or nuclear early antigens. Such patients are thus able to lyse immortalized cells, release nuclear products, and present them to the immune system. Three hypotheses are suggested to explain the lack of antibody to the K antigen: a viral mutation, a failure of immune recognition, or lack of in vivo expression of the antigen due to extensive viral replication. Lack of antibody to one component of EBNA may serve as an objective serological marker for certain patients with chronic EBV infection.